The present invention is in the field of recombinant DNA technology. This invention is directed to a process for amplifying a nucleic acid molecule, and to the molecules, cells, and non-human transgenic animals employed and/or produced through this process.
Assays capable of detecting the presence of a particular nucleic acid molecule in a sample are of substantial importance in forensics, medicine, epidemiology and public health, and in the prediction and diagnosis of disease. Such assays can be used, for example, to identify the causal agent of an infectious disease, to predict the likelihood that an individual will suffer from a genetic disease, to determine the purity of drinking water or milk, or to identify tissue samples. The desire to increase the utility and applicability of such assays is often frustrated by assay sensitivity. Hence, it would be highly desirable to develop more sensitive detection assays.
The usefulness of a detection assay is often limited by the concentration at which a particular target nucleic acid molecule is present in a sample. Thus, methods that are capable of amplifying the concentration of a nucleic acid molecule have been developed as adjuncts to detection assays.
One method for overcoming the sensitivity limitation of nucleic acid concentration is to selectively amplify the nucleic acid molecule whose detection is desired prior to performing the assay. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments in vivo have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, etc.
In many instances in clinical medicine and diagnostics, however, the concentration of a target species in a sample under evaluation is so low that it cannot be readily cloned. To address such situations, methods of in vitro nucleic acid amplification have been developed that employ template directed extension. In such methods, the nucleic acid molecule is used as a template for extension of a nucleic acid primer in a reaction catalyzed by polymerase.
One such template extension method is the xe2x80x9cpolymerase chain reactionxe2x80x9d (xe2x80x9cPCRxe2x80x9d), which is among the most widely used methods of DNAn amplification (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; Saiki, R. et al., U.S. Pat. No. 4,683,194 and Higuchi, R. xe2x80x9cPCR Technology,xe2x80x9d Ehrlich, H. (ed.), Stockton Press, NY, 1989, pp 61-68), which references are incorporated herein by reference).
The polymerase chain reaction can be used to selectively increase the concentration of a nucleic acid molecule even when that molecule has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotides to serve as primers for the template-dependent, polymerase mediated replication of the desired nucleic acid molecule.
The precise nature of the two oligonucleotide primers of the PCR method is critical to the success of the method. As is well known, a molecule of DNA or RNA possesses directionality, which is conferred through the 5xe2x80x2xe2x86x923xe2x80x2 linkage of the sugar-phosphate backbone of the molecule. Two DNA or RNA molecules may be linked together through the formation of a phosphodiester bond between the terminal 5xe2x80x2 phosphate group of one molecule and the terminal 3xe2x80x2 hydroxyl group of the second molecule. Polymerase dependent amplification of a nucleic acid molecule proceeds by the addition of a nucleotide having 5xe2x80x2 phosphate to the 3xe2x80x2 hydroxyl end of a nucleic acid molecule. Thus, the action of a polymerase extends the 3xe2x80x2 end of a nucleic acid molecule. These inherent properties are exploited in the selection of the two oligonucleotide primers of the PCR. The oligonucleotide sequences of the two primers of the PCR method are selected such that they contain sequences identical to, or complementary to, sequences which flank the sequence of the particular nucleic acid molecule whose amplification is desired. More specifically, the nucleotide sequence of the Amplification Primer is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3xe2x80x2 to the sequence of the desired nucleic acid molecule that is to be amplified, whereas the nucleotide sequence of the Target Primer is selected such that it contains a nucleotide sequence identical to one present 5xe2x80x2 to the sequence of the desired nucleic acid molecule that is to be amplified. Both primers possess the 3xe2x80x2 hydroxyl groups which are necessary for enzyme mediated nucleic acid synthesis.
In the polymerase chain reaction, the reaction conditions must be cycled between those conducive to hybridization and nucleic acid polymerization, and those which result in the denaturation of duplex molecules. In the first step of the reaction, the nucleic acid molecules of the sample are transiently heated, and then cooled, in order to denature any double stranded molecules that may be present. The amplification and Target Primers are then added to the sample at a concentration which greatly exceeds that of the desired nucleic acid molecule. When the sample is then incubated under conditions conducive to hybridization and polymerization, the Amplification Primer will hybridize to the nucleic acid molecule of the sample at a position 3xe2x80x2 to the sequence of the desired molecule to be amplified. If the nucleic acid molecule of the sample was initially double stranded, the Target Primer will hybridize to the complementary strand of the nucleic acid molecule at a position 3xe2x80x2 to the sequence of the desired molecule that is the complement of the sequence whose amplification is desired. Upon addition of a polymerase, the 3xe2x80x2 ends of the amplification and (if the nucleic acid molecule was double stranded) Target Primers will be extended. The extension of the Amplification Primer will result in the synthesis of a DNA molecule having the exact sequence of the complement of the desired nucleic acid. Extension of the Target Primer will result in the synthesis of a DNA molecule having the exact sequence of the desired nucleic acid.
The PCR reaction is capable of exponentially amplifying the desired nucleic acid sequences, with a near doubling of the number of molecules having the desired sequence in each cycle. This exponential increase occurs because the extension product of the Amplification Primer contains a sequence which is complementary to a sequence of the Target Primer, and thus can serve as a template for the production of an extension product of the Target Primer. Similarly, the extension product of the Target Primer, of necessity, contain a sequence which is complementary to a sequence of the Amplification Primer, and thus can serve as a template for the production of an extension product of the Amplification Primer. Thus, by permitting cycles of hybridization, polymerization, and denaturation, an exponential increase in the concentration of the desired nucleic acid molecule can be achieved. Reviews of the polymerase chain reaction are provided by Mullis, K. B. (Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki, R. K., et al. (Bio/Technology 3:1008-1012 (1985)); and Mullis, K. B., et al. (Met. Enzymol. 155:335-350 (1987), which references are incorporated herein by reference).
PCR technology is useful in that it can achieve the rapid and extensive amplification of a polynucleotide molecule. However, the method has several salient deficiencies. First, it requires the preparation of two different primers which hybridize to two oligonucleotide sequences of the target sequence flanking the region that is to be amplified. The concentration of the two primers can be rate limiting for the reaction. Although it is not essential that the concentration of the two primers be identical, a disparity between the concentrations of the two primers can greatly reduce the overall yield of the reaction.
A further disadvantage of the PCR reaction is that when two different primers are used, the reaction conditions chosen must be such that both primers xe2x80x9cprimexe2x80x9d with similar efficiency. Since the two primers necessarily have different sequences, this requirement can constrain the choice of primers and require considerable experimentation. Furthermore, if one tries to amplify two different sequences simultaneously using PCR (i.e. using two sets of two primers), the reaction conditions must be optimized for four different primers.
A further disadvantage of PCR is that it requires the thermocycling of the molecules being amplified. Since this thermocycling requirement denatures conventional polymerases, it thus requires the addition of new polymerase at the commencement of each cycle. The requirement for additional polymerase increases the expense of the reaction, and can be avoided only through the use of thermostable polymerases, such as Taq polymerase. Moreover, the thermocycling requirement attenuates the overall rate of amplification because further extension of a primer ceases when the sample is heated to denature double-stranded nucleic acid molecules. Thus, to the extent that the extension of any primer molecule has not been completed prior to the next heating step of the cycle, the rate of amplification is impaired.
Other known nucleic acid amplification procedures include transcription-based amplification systems (Kwoh D. et al., Proc. Natl. Acad Sci. (U.S.A.) 86:1173 (1989); Gingeras T. R. et al., PCT appl. WO 88/10315 (priority: U.S. patent application Ser. Nos. 064,141 and 202,978); Davey, C. et al., European Patent Application Publication No. 329,822; Miller, H. I. et al., PCT appl. WO 89/06700 (priority: U.S. patent application Ser. No. 146,462, filed Jan. 21, 1988)), and xe2x80x9cracexe2x80x9d (Frohman, M. A., In: PCR Protocols: A Guide to Methods and Applications, Academic Press, NY (1990)) and xe2x80x9cone-sided PCRxe2x80x9d (Ohara, O. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86.5673-5677 (1989)).
Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting xe2x80x9cdi-oligonucleotidexe2x80x9d, thereby amplifying the di-oligonucleotide, are also known (Wu, D. Y. et al., Genomics 4:560 (1989)).
An isothermal amplification method has been described in which a restriction endonuclease is used to achieve the amplification of target molecules that contain nucleotide 5xe2x80x2-[a-thio]triphosphates in one strand of a restriction site (Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)).
All of the above amplification procedures depend on the principle that an end-product of a cycle is functionally identical to a starting material. Thus, by repeating cycles, the nucleic acid is amplified exponentially.
Methods that use thermocycling, e.g. PCR or Wu, D. Y. et al., Genomics 4:560 (1989)), have a theoretical maximum increase of product of 2-fold per cycle, because in each cycle a single product is made from each template. In practice, the increase is always lower than 2-fold. Further slowing the amplification is the time spent in changing the temperature. Also adding delay is the need to allow enough time in a cycle for all molecules to have finished a step. Molecules that finish a step quickly must xe2x80x9cwaitxe2x80x9d for their slower counterparts to finish before proceeding to the next step in the cycle; to shorten the cycle time would lead to skipping of one cycle by the xe2x80x9cslowerxe2x80x9d molecules, leading to a lower exponent of amplification.
The present invention concerns a method and in vitro polynucleotide complexes for achieving the amplification of a nucleic acid molecule using a single primer, under isothermal conditions.
In detail, the invention provides a composition for amplifying in vitro a target polynucleotide region of an initial linear nucleic acid molecule, wherein the composition comprises:
(A) a single-stranded first polynucleotide, wherein the polynucleotide (i) contains a polynucleotide region that is complementary in sequence to the target polynucleotide region, and (ii) is a circular polynucleotide or is circularizable when hybridized to the target polynucleotide region in vitro; and
(B) a second polynucleotide comprising the target polynucleotide region.
The present invention particularly concerns the embodiments of such a composition wherein the composition additionally comprise a template-dependent polymerase sufficient to extend a 3xe2x80x2 terminus of a polynucleotide hybridized to the single-stranded first polynucleotide in vitro to thereby produce a template-dependent extension product and wherein the polymerase is additionally capable of causing extension-dependent strand displacement of hybridized polynucleotides.
The present invention particularly concerns the embodiments of such compositions wherein, the single-stranded first polynucleotide is circularizable via the action of a ligase, or is circularizable via the action of a recombinase.
The present invention particularly concerns the embodiments of such compositions wherein, the single-stranded first polynucleotide contains a modified nucleotide, especially a ribonucleotide or a biotinylated nucleotide.
The present invention further concerns a kit for amplifying in vitro a target polynucleotide region of an initial linear nucleic acid molecule, wherein the kit comprises:
(A) a first container containing a single-stranded first polynucleotide, wherein the polynucleotide (i) contains a polynucleotide region that is complementary in sequence to the target polynucleotide region, and (ii) is a circular polynucleotide or is circularizable when hybridized to the target polynucleotide region; and
(B) a second container containing a second polynucleotide comprising the target polynucleotide region.
The present invention particularly concerns the embodiments of such a kit wherein the kit additionally comprise a third container containing a template-dependent polymerase sufficient to extend a 3xe2x80x2 terminus of a polynucleotide hybridized to the single-stranded first polynucleotide in vitro to thereby produce a template-dependent extension product and wherein the polymerase is additionally capable of causing extension-dependent strand displacement of hybridized polynucleotides.
The present invention particularly concerns the embodiments of such kits wherein, the single-stranded first polynucleotide is circularizable via the action of a ligase, or is circularizable via the action of a recombinase.
The present invention particularly concerns the embodiments of such kits wherein, the single-stranded first polynucleotide contains a modified nucleotide, especially a ribonucleotide or a biotinylated nucleotide.